SPECIFIC AIMS. It is clear that freezing can and does destroy the cells and tissues of prostate cancer as well as many other cancers. The use of cryosurgery to treat prostate cancer is growing as evidenced by the inclusion of a chapter on its use in Campbell's Urology, the standard text for urology residents and practitioners [1]. Based on our original NIH R29 supported work, we have demonstrated that vascular mediated injury appears to dominate direct cell injury within both a rodent and human prostate cancer model system [2-4] (and Preliminary Results). Nevertheless, clinically there is recurrence of the disease after 5 years in some cryosurgically treated patients which may be connected with the initial challenge of treating all of the cancer near certain anatomical areas such as the rectum [5] [6] [7]. In order to address the clinical issues of recurrence and control as well as further the acceptance and application of the technique, arguably more work is needed to reproducibly define and, if possible, extend the edge of the cryosurgical lesion in vivo. Our preliminary results in human prostate cancer (LNCaP) grown in a nude mouse show for the first time the capability of destroying all tissue frozen within an iceball by use of an appropriate molecular adjuvant. This dramatic result suggests that we may indeed be capable of both controlling and extending the cryolesion in vivo. Quantitative description of the cell and vascular mechanisms responsible for this destructive enhancement as well as the appropriate molecular adjuvants to achieve this are the topic of this submission. The following hypothesis and specific aims create a framework to study these issues: Hypothesis. Direct cell cryo-injury at the level of the vascular endothelium followed by endothelial cell activation and vascular inflammation determines the extent of the cryolesion in vivo. SA 1 - Establish Conditions for Direct Cell Injury (DCl) in human Endothelial Cells (MVECs) and LNCaP In Vitro and Assess Augmentation by Molecular Adjuvants. SA 2- Establish Thermal Thresholds and Cellular and Molecular Nature of In Vivo Cryo- Injury in a Dorsal Skin Fold Chamber (DSFC) Model. SA3- Establish and Enhance Mechanism(s) of In Vivo Cryoinjury Using Molecular Adjuvants (Direct Cell and Vascular) Within the DSFC System.